Neddylation is a novel therapeutic target for lupus by regulating double negative T cell homeostasis

Systemic lupus erythematosus (SLE), a severe autoimmune disorder, is characterized by systemic inflammatory response, autoantibody accumulation and damage to organs. The dysregulation of double-negative (DN) T cells is considered as a crucial commander during SLE. Neddylation, a significant type of protein post-translational modification (PTM), has been well-proved to regulate T cell-mediated immune response. However, the function of neddylation in SLE is still unknown. Here, we reported that neddylation inactivation with MLN4924, a specific inhibitor of NEDD8-activating enzyme E1 (NAE1), or genetic abrogation of Ube2m in T cells decreased DN T cell accumulation and attenuated murine lupus development. Further investigations revealed that inactivation of neddylation blocked Bim ubiquitination degradation and maintained Bim level in DN T cells, contributing to the apoptosis of the accumulated DN T cells in lupus mice. Then double knockout (KO) lupus-prone mice (Ube2m-/-Bim-/-lpr) were generated and results showed that loss of Bim reduced Ube2m deficiency-induced apoptosis in DN T cells and reversed the alleviated lupus progression. Our findings identified that neddylation inactivation promoted Bim-mediated DN T cell apoptosis and attenuated lupus progression. Clinically, we also found that in SLE patients, the proportion of DN T cells was raised and their apoptosis was reduced. Moreover, compared to healthy groups, SLE patients exhibited decreased Bim levels and elevated Cullin1 neddylation levels. Meantime, the inhibition of neddylation induced Bim-dependent apoptosis of DN T cells isolated from SLE patients. Altogether, our findings provide the direct evidence about the function of neddylation during lupus, suggesting a promising therapeutic approach for this disease.


INTRODUCTION
2][3] Double-negative (CD3 + CD4 -CD8 -, DN) T cells, a unique subset of T cells lacking CD4 and CD8 co-receptors, play a significant role in the pathogenesis of autoimmune diseases, such as SLE. 4 During SLE progress, DN T cells invade into multiple organs, contributing to the loss of tolerance.Besides, DN T cells are able to facilitate B cell differentiation to enhance the generation of autoantibodies.][10] Thus, elucidation of the signaling events mediating the homeostasis of DN T cells could provide new potential therapeutic options for SLE.
Neddylation is a type of protein post-translational modification (PTM).In this process, neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8), an ubiquitin-like protein, binds to substrate proteins to regulate their stability, localization or activity. 11,12Similar to ubiquitination, neddylation is also catalyzed by a three-step enzymatic cascade of NEDD8activating enzyme E1 (NAE1), NEDD8-conjugating enzyme E2 (Ube2m or Ube2f) and substrate-specific NEDD8-E3 ligases (RBX1/ ROC1, RBX2/ROC2, etc.). 13The Cullin family, indispensable parts of Cullin-RING ubiquitin E3 ligases (CRL), is well-characterized as substrates for NEDD8 modification.Cullin neddylation is essential for the ubiquitin ligase activity of CRLs, which transfer the ubiquitin from recruited E2-ubiquitin to protein substrates and finally promote substrate ubiquitination. 14,15t is well established that neddylation modification is a critical PTM in modulating T cell-mediated immune response.7][18][19] Thus, neddylation serves as a critical modulator for T cell functions.However, the precise involvement of neddylation in SLE remains unclear.
In our study, we firstly identified that blockade of neddylation pathway with MLN4924, a pharmacological inhibitor of NAE, 20 significantly attenuated SLE progress with reduced DN T cell number in lupus-prone mice.To explore the function of neddylation pathway in SLE, Ube2m, the NEDD8-conjugating enzyme E2, was specifically knockout (KO) to generate spontaneous lupus-prone mice with Ube2m deficiency in T cells (Ube2m -/-lpr), where the neddylation was inactivated.Results showed that Ube2m deficiency attenuated SLE development.Subsequent experiments revealed that this effect resulted from the decreased number of DN T cells.Mechanism studies identified that inactivation of neddylation impaired Bim ubiquitination degradation and maintained Bim level in DN T cells, which induced the apoptosis of accumulated DN T cells in lupus mice.To confirm that neddylation pathway regulated the homeostasis of DN T cells via Bim, we generated the double KO lupus-prone mice (Ube2m -/-Bim -/-lpr) and found that the deficiency of Bim disrupted the apoptosis of DN T cells for Ube2m deficiency and cannot inhibit the development of lupus.Clinically, we also found that SLE patients exhibited an accumulation of DN T cells with reduced apoptosis.Furthermore, comparing the DN T cells from SLE groups with the healthy control, we discovered that the neddylation level of Cullin1 was enhanced while Bim level was decreased.Further research showed that neddylation inhibition with MLN4924 facilitated Bim-dependent apoptosis in DN T cells isolated from SLE groups.Our results confirmed that neddylation is necessary for the homeostasis of DN T cells.Neddylation inhibition initiates Bim-mediated mitochondrial apoptosis and restores the disordered immune tolerance in SLE.These results highlight the importance of neddylation pathway for SLE and suggest a novel therapeutic intervention of lupus via targeting neddylation pathway in DN T cells.

Inhibition of neddylation attenuated lupus progression in MRL/ lpr mice
To investigate the function of neddylation during lupus, female MRL/lpr mice with the Fas gene mutation were used.From about 12 weeks of age, MRL/lpr mice exhibit the expansion of lymphocytes and spontaneously develop SLE-like symptoms resembling human SLE. 10 We then treated MRL/MpJ and MRL/lpr group with DMSO or MLN4924 (a specific inhibitor of neddylation) every 3rd day from 12 weeks to 20 weeks of age (Fig. 1a).Firstly, we found that treatment of MLN4924 remarkably inhibited the neddylation of Cullin1 in splenocytes of MRL/lpr mice (Fig. 1b), suggesting that MLN4924 indeed suppressed neddylation pathway in vivo.Notably, MRL/lpr mice treated with MLN4924 showed significantly higher survival rates than controls (Fig. 1c).Besides, MLN4924 treatment resulted in a significant reduction in spleen size as evidenced by a decreased spleen index (spleen weight/ body weight ratio) (Fig. 1d) and decreased splenocyte number in MRL/lpr mice (Fig. 1e).Additionally, we assessed the levels of serum IgG and anti-dsDNA antibodies, which are important diagnostic markers of lupus. 21We found that MLN4924 prominently reduced IgG (Fig. 1f) and anti-dsDNA (Fig. 1g) antibody levels in MRL/lpr group (Fig. 1f, g).To assess the impact of MLN4924 on kidney function, urine samples were obtained and the levels of urinary total protein, albumin and creatinine were quantified.We noticed that MRL/lpr mice administrated with MLN4924 exhibited a significantly decrease in total protein (Fig. 1h) and albumin/ creatinine ratio (Fig. 1i).Histological analysis of kidney sections stained with PAS revealed that MLN4924 markedly reduced the crescent glomerulonephritis in MRL/lpr group (Fig. 1j), indicating a protective role of MLN4924 in renal function.Serum cytokine levels were evaluated and results indicated that the production of IL-6, IL-17, TNF-α and IFN-γ was inhibited in MRL/lpr group with MLN4924 administration while IL-1β and IL-10 level remained unchanged (Fig. 1k).Together, our data suggest that MLN4924 treatment attenuates lupus symptoms as exemplified by increased survival rate, reduced splenomegaly and autoantibody production, ameliorated renal function, and suppressed inflammatory cytokine levels.
MLN4924 significantly reduced the number of DN T cells in MRL/ lpr mice As indicated in Fig. 1e, splenic cell accumulation was considerably reduced for MLN4924 treatment.Thus, we investigated the impact of MLN4924 on splenic cells by examining the T and B cell proportions.Flow cytometry analysis revealed a significant decrease of the percentage and total count of T cells in MLN4924-administered MRL/lpr mice compared to DMSOtreated mice (Fig. 2a-c) while the proportion and number of B cells remained unchanged (Fig. 2a, d, e).Further analysis showed a moderate reduction in DN T cell percentage but a significant decrease in DN T cell number for MLN4924 treatment (Fig. 2f-h), indicating that inhibition of neddylation suppressed the accumulation of DN T cells in MRL/lpr mice.

Deficiency of Ube2m in T cells attenuated SLE development
To thoroughly assess the role of neddylation in SLE, LckcreUbe2m fl/ fl MRL.Fas lpr mice (termed Ube2m -/-lpr mice) were generated according to the procedure in Supplementary Fig. 1a.Ube2m -/-lpr mice showed Ube2m-specific deletion in T cells and spontaneously developed lupus.To assess deletion efficiency, Ube2m protein levels were monitored and results showed a clear reduction in purified T cells of Ube2m -/-lpr mice, while Ube2f, another E2 NEDD8 conjugating enzyme, remains unchanged (Supplementary Fig. 1b), which suggested the specific deletion of Ube2m in Ube2m -/-mice.
Both the percentages and numbers of different T cell subsets from thymus of these mice were analyzed and results showed that Ube2m -/-and Ube2m -/-lpr mice exhibited normal T cell development in thymus, including normal DN T cells, DP cells, CD4 + and CD8 + T cells (Supplementary Fig. 2a-e).Based on the presence of the specific surface markers, CD25 and CD44, the DN stages have been classified as DN1, DN2, DN3 and DN4. 22Therefore, we further characterized DN populations according to the expression of CD25 and CD44 to explore whether Ube2m deficiency in T cells affects the development of DN T cells.As shown in Supplementary Fig. 2c, f, g, T cells lacking Ube2m exhibited unchanged DN T cells.The normal development of T cells in thymus of Ube2m KO mice allowed further investigation of the effects of Ube2m deficiency on the pathogenesis of lupus.As shown in Fig. 3a-c, Ube2m -/-lpr mice showed dramatically reduced splenomegaly (Fig. 3a, b) and decreased number of splenocytes (Fig. 3c) compared with WTlpr at 8 months.Moreover, the serum IgG and anti-dsDNA antibody production was notably abated (Fig. 3d, e).Furthermore, we also found that deficiency of Ube2m ameliorated lupus nephritis, including lessened total protein (Fig. 3f) and albumin/ creatinine ratio (Fig. 3g), restored renal structures (Fig. 3h, i).In addition, Ube2m -/-lpr mice showed markedly reduced levels of inflammatory cytokines (Fig. 3j).Collectively, these results clearly showed that neddylation inactivation in T cells significantly attenuated lupus progression.

Loss of Ube2m promoted DN T cell apoptosis
In vivo evidence establishing the crucial role of Ube2m in lupus development, we further explored how Ube2m regulated lupus progress.Firstly, we measured the number and proportion of T cells in spleen and results revealed that loss of Ube2m prominently blocked total T cell accumulation both in spleen (Fig. 4a-c) and peripheral blood (Supplementary Fig. 3a, b).Further research showed that DN T cell accumulation in spleen (Fig. 4d-f) and peripheral blood (Supplementary Fig. 3c, d) were significantly inhibited in Ube2m -/-lpr.Our previous results (Supplementary Fig. 2c-g) have demonstrated that the development of DN T cells in thymus is not affected by Ube2m KO.Therefore, we then detected the influence of Ube2m on DN T cells in spleens.In view of the deficit of Fas-triggered apoptosis in lpr mice, 23 we firstly measured the level of T cell apoptosis in spleens.Consistent with our above-mentioned phenotype, the apoptosis of DN T cells was blocked in WTlpr mice compared to WT mice, while disruption of Ube2m restored the normal apoptosis of T cells, especially DN T cells (Fig. 4g-i).Similar results were found in peripheral blood (Supplementary Fig. 4a-c).Consistently, we also observed that loss of Ube2m significantly promoted DN T cell apoptosis (Fig. 4j, k) and attenuated disease progression (Supplementary Fig. 5) in pristane-induced lupus mice.Then, JC-1 staining assay indicated that Ube2m-deleted DN T cells exhibited reduced MMP, as evaluated by the ratio of JC-1 aggregates to monomer (Fig. 4l), which indicated increased apoptosis.Consistently, we also found that Ube2m deficiency resulted in an elevated cleaved-caspase 3 level (Fig. 4m) in DN T cells, which is required for cell apoptosis.Besides, the excessive T cell proliferation is another striking abnormality of lpr mice 24 and our results showed that Ube2m deficiency inhibited total T cell proliferation in vivo (Supplementary Fig. 6a, b).Notably, loss of Ube2m mainly restrained proliferation of CD4 + and CD8 + T cells while having minimal effect on DN T cell proliferation (Supplementary Fig. 6a, c), which suggested differential functions of Ube2m in different T cell subsets.These findings demonstrated the essential role of Ube2m in the survival of DN T cells, and deletion of Ube2m facilitated DN T cell apoptosis in lupus mice.

Neddylation inactivation upregulated Bim level by inhibiting Bim ubiquitination degradation
We have shown that loss of Ube2m promoted DN T cell apoptosis and contributed to alleviated lupus development (Figs. 3 and 4), while the mechanism remained unknown.Therefore, DN T cells were sorted and an unbiased quantitative proteomic analysis was conducted to screen for the differential protein between WTlpr and Ube2m -/-lpr mice.Results indicated that the protein Bim (encoded by the Bcl2l11 gene) showed the most significant increase among the apoptosis-related proteins for Ube2m deficiency (Fig. 5a).Next, the mRNA level of Bim was measured and results indicated that the transcriptional expression of Bim remained unchanged for loss of Ube2m (Fig. 5b, c).Based on our data, we speculated that neddylation pathway regulated Bim degradation.Consequently, MG-132, a proteasome inhibitor, was used to treat DN T cells.Results indicated that Bim was accumulated upon MG-132 treatment (Fig. 5d), suggesting that Bim degradation was proteasome-dependent. Previous studies have proven that Bim ubiquitination degradation is dependent on the neddylation of Cullin1. 25,26As a result, the neddylation of Cullin1 was detected and results showed that Cullin1 neddylation was at a lower level in normal mice and remained little change for Ube2m deficiency, resulting in unchanged Bim protein level in DN T cells under normal physiological condition (Fig. 5e).However, we found that Ube2m expression and the neddylation of Cullin1 was significantly increased in lupus conditions (Fig. 5e), suggesting that the neddylation pathway activation was necessary for DN T cell abnormal survival in lupus progression.Then, loss of Ube2m prominently blocked the neddylation of Cullin1, contributing to the Bim protein accumulation (Fig. 5e) and finally promoting DN T cell apoptosis in lupus mice (Fig. 4g, i).Furthermore, Co-IP assay revealed that the ubiquitination degradation of Bim was indeed remarkably inhibited for Ube2m deletion in DN T cells (Fig. 5f).In vitro results also showed that neddylation inactivation with MLN4924 promoted DN T cell apoptosis (Fig. 5g, h), with elevated Bim and cleaved-caspase 3 protein levels (Fig. 5i).Further Co-IP assay also indicated that MLN4924 treatment disrupted Bim ubiquitination degradation, contributing to the accumulation of Bim (Fig. 5j).Taken together, we demonstrated that neddylation inactivation impairs Bim ubiquitination degradation and maintains Bim level, ultimately promoting the apoptosis of DN T cells in lupus mice.

Loss of Bim reduced Ube2m deficiency-induced apoptosis in DN T cells and reversed the alleviated lupus progression
According to our obtained findings, we hypothesized that Bim is a pivotal protein downstream mediated by neddylation pathway, responsible for DN T cell homeostasis.To further test our hypothesis, LckcreBim fl/fl MRL.Fas lpr (termed Bim -/-lpr) and LckcreU-be2m fl/fl Bim fl/fl MRL.Fas lpr (termed Ube2m -/-Bim -/-lpr) mice were generated.Firstly, we found that deletion of Bim in T cells led to an increase both in splenic and T cell numbers (Fig. 6a, b).Further analysis revealed that there was a dramatic increase in the proportion and number of DN T cells (Fig. 6c-e) due to reduced apoptosis (Fig. 6f-h) in Bim -/-lpr compared with WTlpr mice, suggesting a significant function of Bim in DN T cell homeostasis.Then, we also found that lack of Bim blocked the increased apoptosis of DN T cells resulted from Ube2m deficiency (Fig. 6f, h), contributing to more DN T cells in Ube2m -/-Bim -/-lpr mice compared with Ube2m -/-lpr mice (Fig. 6c-e).Moreover, when Bim is lost, the lack of Ube2m has little effects on the apoptosis and number of DN T cells (Fig. 6c-h).Thus, Bim served as a key downstream protein of neddylation pathway to regulate DN T cell apoptosis.In addition, Ube2m -/-Bim -/-lpr mice showed reduced T cell (Fig. 6b) and CD4 + T cell number (Fig. 6e) with unchanged T cell and CD4 + T cell apoptosis compared with Bim -/-lpr mice (Fig. 6f-h), which suggested that there are some Bim-independent mechanisms to regulate CD4 + T cell homeostasis.
Considering the important function of DN T cells in lupus progress, lupus syndrome of these mice was also detected in Supplementary Fig. 7. Consistently, Bim -/-lpr mice exhibited the most severe lupus syndrome, including splenomegaly (Supplementary Fig. 7a), the accumulation of autoantibodies (Supplementary Fig. 7b, c), renal destruction (Supplementary Fig. 7d-g) as well as increased inflammatory cytokine level (Supplementary Fig. 7 h).Meanwhile, compared with Bim -/-lpr mice, Ube2m -/-Bim -/-lpr mice also showed slightly remission on lupus development (Supplementary Fig. 7), indicating a Bim-independent manner of Ube2m on lupus development.In addition, we also found that the attenuated lupus phenotypes in Ube2m KO mice were mainly reversed in Ube2m -/-Bim -/-lpr mice (Supplementary Fig. 7).Collectively, these evidences clearly identified that Bim is an indispensable downstream protein governed by neddylation pathway to regulate DN T cell apoptosis, which mediates the development of lupus.

Inhibition of neddylation pathway promoted Bim-dependent DN T cell apoptosis in SLE patients
We have verified that neddylation inactivation induced Bimdependent DN T cell apoptosis in lupus-prone mice.However, the impact of neddylation inactivation on DN T cells derived from SLE patients remains unknown.Peripheral blood samples were collected from both healthy individuals and patients with active SLE.Firstly, we observed that patients with SLE showed a higher percentage of total DN T cells with lower apoptosis in comparison to the healthy group (Fig. 7a-c).The subsequent correlation analysis revealed a positive association between the percentage of DN T cells and SLE-DAI scores (Fig. 7d), then a negative association between the apoptosis proportion of DN T cells and SLE-DAI scores (Fig. 7e).Further exploration showed that Bim protein level was reduced, while the neddylation of Cullin1 was increased in DN T cells of SLE group (Fig. 7f).On this basis, MLN4924 was used to treat the PBMC isolated from SLE patients and results revealed that neddylation inhibition declined the DN T cell percentage (Fig. 7g, h) and promoted the apoptosis of DN T cells (Fig. 7i, j).Consistently, we found the Bim level was upregulated (Fig. 7k) upon neddylation inhibition.These clinical data identified that neddylation inactivation promotes the Bim-dependent DN T cell apoptosis, thus contributing to the reduction of DN T cells.Our data suggested In conclusion, our research uncovers the role of neddylation pathway in DN T cell homeostasis (Fig. 8), thereby providing a novel treatment approach for SLE.

DISCUSSION
SLE is an autoimmune disorder and the underlying causes remains mostly unknown.The abnormal accumulation of DN T cells, which led to the peripheral tolerance defects and systemic damage, is a key pathogenesis of SLE. 4,27Thus, it is necessary to elucidate the mechanisms controlling the homeostasis of DN T cells during SLE.Here, we functionally characterized the unique role of neddylation in DN T cell homeostasis with a specific inhibitor of neddylation and a genetic approach.Neddylation inactivation promoted the apoptosis of DN T cells via stabilizing the Bim level and contributed to SLE remission.These findings underscore the crucial role of neddylation in DN T cell functions and indicate neddylation as a potential target for SLE therapy.9][30] In addition, ongoing investigations implicate neddylation is necessary for the regulation of immune cell functions and involved in the pathogenesis of related immune disorders. 31][34] Neddylation is also required for the IFN production against RNA viral infection in myeloid DCs. 357][38] However, the role of neddylation in DN T cells remains to be explored.
The homeostasis of DN T cells was involved in the development of SLE. 4,10DN T cell apoptosis and timely clearance are critical for immune tolerance, which avoided the release of autoantigen and the induction of autoimmunity.0][41] Therefore, the normal apoptosis and clearance of DN T cells are beneficial for lupus remission.Bim, belonging to the Bcl-2 family, is a crucial BH3 protein promoting cell intrinsic apoptosis. 42In mammalian, two main pathways govern the initiation of apoptosis.One is the extrinsic pathway (or death receptor pathway), triggered by ligand engagement of cell surface death receptors such as Fas.Another is intrinsic apoptotic pathway, which is mediated by Bcl-2 protein family. 435][46] Loss of either Fas or Bim resulted in marked lymphadenopathy and splenomegaly, and deficiency of Bim in MRL/ lpr mice developed more extreme lymphadenopathy, indicating that Fas signaling and Bim showed overlapping but non-redundant roles in lymphadenopathy and splenomegaly. 47,48However, the precise role of Bim in DN T cells and the underlying regulatory mechanism governing Bim homeostasis remained elusive.In our study, we found that neddylation inactivation observably reduced DN T cell accumulation in lupus mice with upregulated Bim protein level and normal transcription of Bim.MG-132 treatment suggested that Bim degradation was mediated by ubiquitin-proteasome system.Previous studies have proven that Bim protein was the substrates of  25,26,49 Therefore, we also detected the Bim ubiquitination degradation and found that the Bim degradation was significantly inhibited accompanied with impaired Cullin1 neddylation in DN T cells when neddylation was inactivated.However, Bim accumulation and Cullin1 neddylation inhibition of DN T cells were only found in lupus mice rather than in non-lupus group.Our data indicated that Ube2m specifically regulates Bim in SLE group.In normal physiological condition, the remaining low level of Ube2m after Cre driven deletion appears to be sufficient to maintain the basal level of Cullin1 neddylation.However, under lupus conditions, Ube2m was significantly induced to ensure sufficient Cullin1 neddylation to activate CRL1, which is consistent with the previous work showing that Ube2m is a stress inducible protein. 50And Ube2m deletion failed to provide enough Cullin1 neddylation, leading to CRL1 inactivation and subsequent Bim accumulation to induce apoptosis in lupus groups.To further verify the involvement of Bim in neddylation-mediated DN T cell apoptosis, Ube2m -/-Bim -/-lpr mice were generated and results showed that when Bim was deleted, neddylation inactivation failed to promote DN T cell apoptosis, suggesting that neddylation pathway mediated DN T cell apoptosis via regulating Bim protein degradation.
In our research, we also found that neddylation function has heterogeneity in different T cell subsets.Our data revealed that neddylation inactivation also blocked Bim ubiquitination and increased Bim protein level in CD4 + and CD8 + T cells similar with DN T cells (Supplementary Fig. 8a-d).However, the susceptibility of DN T cells to Bim-induced apoptosis was found to be the highest (Fig. 4g-i, Fig. 5g, h and Supplementary Fig. 8e-h).It has been well demonstrated that cellular metabolism can influence cell sensitivity to apoptosis. 51,52We hypothesized that the heterogeneity of neddylation functions in different T cells depended on their distinct metabolic profiles.More research is necessary to comprehensively elucidate the metabolic modes of different T cells as well as the relationship between apoptosis and metabolic modes during lupus.Besides, we found that neddylation inactivation notably suppressed the proliferation of CD4 + T cells and blocked the differentiation of T follicular helper (Tfh) cells in MRL/lpr mice and pristane-induced lupus models (data not shown).CD4 + T cells, such as Th1, Tfh and Th17 cells also contribute to tissue inflammation and autoantibody production, promoting the pathogenesis of SLE. 2,53,54These changes also explained why Ube2m -/-Bim -/-lpr mice exhibited slightly reduced T cell number and attenuated lupus symptoms compared with Bim -/-lpr mice.However, the mechanism of neddylation pathway in regulating the function of CD4 + T cells during SLE progression requires further investigation.
In summary, we have identified a significant role of neddylation in maintaining DN T cell homeostasis and mediating SLE progression.Suppression of neddylation with MLN4924 or genetic abrogation of Ube2m significantly ameliorated the progression of SLE via reconstructing the normal apoptosis of DN T cells.Further experiments indicated that the interception of apoptosis in lupus mice was indeed recovered for neddylation inactivation, which resulted from the inhibition of Bim ubiquitination and the rise of Bim-mediated apoptosis.The clinical data also showed that SLE patients displayed accumulated DN T cells with defects in apoptosis while neddylation inhibition promoted the apoptosis of DN T cells via up-regulating Bim protein level.The findings enhance our comprehension about the pathogenesis of SLE and suggest that neddylation pathway might be a promising target for SLE therapy.

Mice
We purchased MRL/lpr and the control MRL/Mpj mice (female, 6 weeks) from SLRC laboratory Animal centre.From 12 weeks to 20 weeks, MLN4924 (15 mg/kg) (MCE) or DMSO were intraperitoneally injected into these mice every 3rd day.The mice status and the deaths were observed and recorded daily.The mice were narcotized at 20 weeks in order to obtain serum and blood for flow cytometry.After that, the mice were sacrificed.Kidneys and spleens were obtained.The weight of spleens was evaluated and the total count of splenic cells was determined using the CEDEX XS system (Roche).The Ube2m fl/fl mice were generous gifts from Yi Sun (Zhejiang University) and C57BL/6MRL.Fas lpr mice were generous gifts from Jun Yan (University of Louisville).Lckcre mice and Bim fl/fl mice were bought from Shanghai Model Organisms.LckcreUbe2m fl/fl mice (termed Ube2m -/-mice) were generated by crossing Lckcre mice with Ube2m fl/fl mice.Then these mice were crossed with C57BL/6MRL.Fas lpr mice to acquire spontaneous lupus-prone mice with Ube2m deficiency (LckcreUbe2m fl/fl MRL.Fas lpr , termed Ube2m -/-lpr mice) in T cells.LckcreUbe2m +/+ MRL.Fas lpr or Ube2m fl/fl MRL.Fas lpr mice were as control (termed WTlpr mice).LckcreBim fl/fl mice (termed Bim -/-mice) were generated by crossing Lckcre mice with Bim fl/fl mice.Then these mice were crossed with C57BL/6MRL.Fas lpr mice to acquire LckcreBim fl/fl MRL.Fas lpr mice, termed Bim -/-lpr.LckcreUbe2m fl/fl Bim fl/fl MRL.Fas lpr mice, termed Ube2m -/-Bim -/-lpr, were generated by crossing LckcreUbe2m fl/fl MRL.Fas lpr with Bim fl/fl MRL.Fas lpr mice.LckcreUbe2m +/+ Bim +/+ MRL.Fas lpr or Ube2m fl/fl Bim fl/fl MRL.Fas lpr mice were as control (collectively called WTlpr mice).Thymus index (thymus weight to mouse weight ratio) and the number of cells in thymus were calculated and T cell subsets in thymus were examined via flow cytometry from 8-week mice.At 8 months, the blood was obtained for flow cytometry and serum was collected.Subsequently, the mice were euthanized, and their kidneys and spleens were obtained for subsequent experimental analysis.For pristane-induced lupus, WT and Ube2m -/-female mice (2 months) were administered with 0.5 ml of pristane or PBS via intraperitoneal injection.At 8 months, serum was collected and the mice were euthanized.Spleens and kidneys were obtained for following experiments.
All animal experiments in the study underwent review and were approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University.

Human samples
We collected peripheral blood samples with EDTA anticoagulant from normal and active SLE subjects.The patients met the classification criteria of the 2012 Systemic Lupus International Collaborating Clinics (SLICC) and were subjected to full history taking, thorough clinical and laboratory investigations. 55Then SLE disease activity index (SLE-DAI) was employed to determine the disease activity level.Healthy people were age and gendermatched individuals without underlying medical conditions.The research received approval from the Medical Ethics Committee of Zhejiang Chinese Medical University (2021-KL-1230-1) and consent informed consents have been provided freely by all participants.

Detection of autoantibodies
The concentrations of total IgG and anti-dsDNA in serum were measured following the guidelines provided by the manufacturer (Multisciences Biotech Co., Ltd).
The assessment of renal function Fresh urine samples were manually harvested prior to sacrificing the mice.Then urine protein, albumin and creatinine were determined with corresponding kits following the instructions provided by Dia Sys Diagnostic Systems GmbH.The periodic acid-Schiff (PAS) and hematoxylin eosin (H&E) staining of renal histology as well as the pathological scores were performed according to our previous study. 56o-Plex cytokine assay Serum cytokines including IL-1β, IL-6, IL-10, IL-17, TNF-α and IFN-γ were measured using a Bio-Plex Pro Mouse Cytokine 6-plex panel (Bio-Rad) in accordance with the manufacturer's protocols.
Purification of DN T cells and T cells DN T cells of spleens from mice were isolated using fluorescent cell sorting via BD FACSAria (BD Biosciences) by staining cells with anti-CD3 (PE), anti-CD4 (PE-CY7), anti-CD8 (APC) antibodies.CD4 + and CD8 + T cells from mice were obtained from spleens with corresponding Mouse T Cell Isolation Kits following the protocols provided by manufacturer (Stem Cell Technologies Inc).DN T cells of human peripheral blood were also isolated using a human DN T cell isolation kit (Miltenyi Biotec).

4D label free quantitative proteomic analysis
The 4D label-free quantitative proteomic analysis was conducted by Jingjie PTM BioLabs including protein extraction, trypsin Signal Transduction and Targeted Therapy (2024) 9:18

Fig. 2
Fig. 2 Reduced T cell accumulation in MLN4924-treated MRL/lpr mice.a Proportion of T and B cells in spleens was evaluated by flow cytometry.n = 4/group.b-e The percentage and absolute numbers of T cells and B cells were quantified and calculated statistically based on the analysis of flow cytometry.n = 4/group.*P < 0.05, **P < 0.01, ***P < 0.001.f CD3 + T cell subsets in spleens were analyzed using flow cytometry.n = 4/group.g, h Percentage and total count of T cell subsets were quantified and calculated based on the flow cytometric analysis, n = 4/group.*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4
Fig. 4 Increased DN T cells apoptosis in Ube2m KO lupus-prone mice.a Proportion of T cells in spleens was analyzed by flow cytometry.n = 4 or 5/group.b, c The percentage and number of T cells were calculated based on the data of flow cytometry.n = 4 or 5/group.*P < 0.05.d CD3 + T cell subsets of spleens were analyzed via flow cytometry.n = 4 or 5/group.e, f The percentage and count of T cell subsets were determined using flow cytometry data.n = 4 or 5/group.*P < 0.05, **P < 0.01, ***P < 0.001.g The early apoptosis (Annexin V + /7-AAD -) of total T cells, DN T cells, CD4 + and CD8 + T cells was evaluated with flow cytometry.n = 4 or 5/group.h, i The apoptosis of total T cells and T cell subsets was quantified based on the analysis of flow cytometry.n = 4 or 5/group.*P < 0.05, **P < 0.01, ***P < 0.001.j DN T cell apoptosis (Annexin V + /PI -) in spleens from pristine-induced mice was analyzed with flow cytometry.n = 4 or 5/group.k DN T cell apoptosis in spleens of pristine-induced mice was quantified based on the analysis of flow cytometry.n = 4 or 5/group.*P < 0.05.l MMP was assessed using JC-1 assay to indicate the apoptosis of DN T cells.n = 5/group.***P < 0.001.m Cleaved-caspase 3 was detected via immunoblotting assay to indicate the apoptosis of DN T cells.n = 3/group

Fig. 5
Fig. 5 Bim level was increased with impaired ubiquitination degradation in DN T cells for neddylation inhibition.a The proteins annotated with apoptosis were selected and ranked based on the ratio value from the proteomic results of DN T cells.Red dots represented proteins significantly upregulated (FC＞ 1.5, P < 0.05); Blue dots represented proteins significantly downregulated (FC < 0.667, P < 0.05).n = 4/group.b, c The mRNA levels of Ube2m and Bim in DN T cells were evaluated via real-time PCR.n = 3/group.*P < 0.05, **P < 0.01.d Immunoblotting assay and densitometric analysis evaluated the protein level of Bim in DN T cells treated with MG-132.*P < 0.05.e Western blot assay was conducted to determine the level of Cullin1 neddylation, Ube2m and Bim in DN T cells.One band represented one mouse.n = 3. f Ubiquitination degradation of Bim in DN T cells was measured via Co-IP assay.Data were representative of three independent experiments.g The apoptosis of DN T cells treated with MLN4924 were detected via flow cytometry (Annexin V + /7-AAD -).h The apoptosis of DN T cells treated with MLN4924 was quantified based on the analysis of flow cytometry.n = 3/group.*P < 0.05.i Immunoblotting assay and densitometric analysis showed the level of Bim and Cleaved-caspase3 in MLN4924 treated-DN T cells.*P < 0.05.j Ubiquitination degradation of Bim in MLN4924-treated DN T cells was measured via Co-IP assay.Data were representative of three independent experiments

Fig. 7
Fig. 7 Neddylation inhibition induced the Bim-dependent DN T cell apoptosis isolated from SLE patients.a The percentage and the apoptosis of DN T cells from both SLE groups and healthy individuals were assessed using flow cytometry.n = 9/group.b DN T cell percentage was quantified based on the results of flow cytometry.n = 9/group.*P < 0.05.c The proportion of DN T cell apoptosis were quantified based on the results of flow cytometry.n = 9/group.*P < 0.05.d Analysis of the correlation between the SLE-DAI scores and the percentage of DN T cells in SLE groups.n = 9/group.R = 0.8951, **P < 0.01.e Analysis of the correlation between the SLE-DAI scores and the apoptosis percentage of DN T cells in SLE groups.n = 9/group.R = −0.8536,**P < 0.01.f Immunoblotting assay and densitometric analysis evaluated the protein level of Bim and Cullin1 neddylation in DN T cells isolated from healthy and SLE group.In HC group, each band was a combination of two samples, while in SLE group, one band represented one samples.**P < 0.01.g, h The PBMC isolated from SLE patients was incubated with MLN4924 (0.5 μM) and the proportion of DN T cells were analyzed via flow cytometry.The results were quantified based on the data of flow cytometry.n = 3/group.*P < 0.05.i, j The PBMC isolated from SLE patients was treated with MLN4924 (0.5 μM) and the apoptosis of DN T cells were assessed via flow cytometry (Annexin V + /PI -).The results were quantified based on the flow cytometry data.n = 3/group.**P < 0.01.k Immunoblotting assay evaluated the protein level of Bim and Cullin1 neddylation in DN T cells isolated from SLE patients treated with MLN4924 (0.5 μM).Data were representative of three independent experiments

Fig. 8
Fig. 8 Schematic illustration showed the mechanism of neddylation pathway in lupus treatment.DN T cells enrichment results in the destruction of immune tolerance with the mass secretion of IFN-γ and IL-17, autoantibody accumulation and the damage of multi-organs, leading to the development of lupus.Inactivation of neddylation pathway (MLN4924 treatment or Ube2m KO in T cells) impaired the neddylation of Cullin1 and then interrupted the ubiquitination of Bim, contributing to the Bim-dependent DN T cell apoptosis, which restores the immune tolerance and finally alleviates lupus symptoms.This figure was created by Figdraw (www.figdraw.com)